1. Field of the Invention
The present invention relates generally to the fields of biochemical endocrinology and anti-neoplastic pharmacology. More specifically, the present invention relates to novel GnRH analogues having antitumour effects and pharmaceutical compositions thereof.
2. Description of the Related Art
It is known that this factor of hypothalamic origin (a peptide hormone built up of 10 amino acids) is responsible for the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). A number of agonistic and antagonistic analogues of GnRHs proved to be not only useful for the successful influencing of processes of the reproduction biology but also suitable as antitumour drugs.
GnRH analogues can exert their antitumour effect not only through chemical castration but also in a selective way by directly acting on the tumour cells. The presence of receptor(s) specifically binding GnRH or GnRH analogues, respectively, as well as that of GnRH-mRNA were shown in cell cultures of cancers of human mammary, prostate, ovary and pancreas. Furthermore, the in vitro proliferation-inhibiting action of GnRH analogues was proven on the same cell lines. The specific binding of tritium-labelled D-Phe6-GnRH(1-9)-ethylamide (OVURELIN), a human GnRH superagonist, to cells of MCF-7 and MDA-MB-231 human mammary carcinoma cell lines was demonstrated in experiments. These results confirm the presence of receptor(s) specifically binding GnRH analogues, which is a fundamental condition for development of a direct effect. Similarly, D-Trp6-hGnRH (DECAPEPTYL), an agonistic analogue brought into the therapeutical practice, proved to possess a receptor on the MDA-MB-231 tumour cell line and a direct growth-inhibiting effect on the human mammary tumour cell line mentioned above.
Based on the effective concentration it can be supposed that low-affinity binding site or sites may play an important role in the development of direct antitumour effect. According to the literature the direct antitumour action of GnRH analogues occurs only at relatively high peptide concentrations (10xe2x88x926-10xe2x88x925 M). This pharmacological effect can be achieved only in the case when the active molecule is present in the body not only in a high concentration but also for a long time. For a long time, GnRH was not believed to be a species-specific hormone; it has become known as late as in the early 80""s that the structure of gonadoliberin of some fish and bird species, respectively, is different from that of the mammals. In comparison to the mammalian GnRH, the structure of fish- and bird-specific GnRH, respectively, differs in the amino acid position(s) 7 and/or 8. In relation to the release of LH and FSH, respectively, of mammals, the analogues of chicken GnRH or salmon GnRH are not hyperactive and therefore, they do not desensitize the gonadotropic cells of hypophysis in the corresponding dose range. The composition of mammalian, e.g. human, GnRH is as follows: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO:1).
In 1993, researchers isolated and synthesized the Lamprey-III-GnRH decapeptide from lamprey (Petromyzon marinus) (pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2 (SEQ ID NO:2)). This Lamprey-III-GnRH (herein below GnRH-III) exerts a significant tumour growth-inhibiting effect on human mammary tumour cell lines. Simultaneously, on investigating the endocrinological effect of GnRH-III on rat hypophysis by using the superfusion method it has been found that the LH-releasing effect of this hormone is about thousand times weaker than that of the human GnRH. In the course of in vivo investigations it has been found that, during a prolonged treatment for three cycles, it did not inhibit the ovulation of female rats even in high doses; therefore, it did not induce desensitisation and chemical castration. In relation thereto, a xe2x80x9cflair upxe2x80x9d tumour growth occurring at the beginning of treatment did not appear on the tumour-bearing animals in contrast to other known human hormone analogues acting through the same mechanism of action. Thus, GnRH-III is a selective, highly active antitumour compound.
During the therapeutical use of peptide hormones and their synthetic analogues, a frequent demand is to retain the amount of the pharmacologically active molecule at a high and steady level. Thus, e.g. several agonistic and antagonistic synthetic analogues of gonadoliberin (GnRH), a peptide hormone built up from ten amino acids stimulating the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the hypophysis, proved to be useful not only for successfully influencing processes of reproduction biology but also to provide the possibility of use as antitumour drugs. Depending on the way of use, GnRH and its analogues are able to stimulate or inhibit the secretion of gonadotropin. On carrying out a treatment repeatedly or continuously for a prolonged time with GnRH analogues, a so-called pharmacological gonadectomy is induced as a result of a desensitizing effect exerted on the hypophysis by a pronounced reduction of release of gonadotropin and steroids. Gonadectomy is a required therapeutical intervention in the treatment of steroid-dependent gonadal disease-entities. Simultaneously, gonadectomy is reversible and does not psychically disturb the patient. These analogues proved to be effective in the therapy of prostate, mamma and endometrium carcinomas and even cancers of pancreas and hypophysis on the basis of most recent data. Synthetic-GnRH antagonists are derivatives competitively inhibiting the native hormone. In the case of potent antagonists the amino acids in positions 1 to 3, 6 and 10 are usually exchanged for non-coded amino acids, e.g. of D-configuration. The anti-ovulatory effect of GnRH antagonists is well known. The GnRH analogues may exert their antitumour action not only through chemical castration but also through a direct effect on the tumour cells. Eidne et al. [J. Clin. Endocrinol. Metab. 64, 423-432 (1987)] demonstrated the presence of low-affinity binding sites in the cell cultures of MCF-7 and MDA-MB-231 human mammary tumours; based on the direct inhibitory effect of antagonistic analogues they concluded that GnRH behaves as an autocrine regulatory factor in mammary carcinoma cells. Results most recently published in the literature confirm the expression of GnRH gene in mammary carcinoma cells, which likely occurs during lactation and malignant transformation [Harris et al.: Cancer Res. 51, 2577-2581 (1991)]. The presence in the neoplastic tissue of GnRH-binding sites supports that GnRH has very probably an immediate palliative effect in the therapy of patients suffering from mammary carcinoma. Human mammary carcinoma cell lines or xenografts developed therefrom are useful in vitro and in vivo model systems for demonstrating the direct action of GnRH. GnRH agonists, when administered alone in substance form, are proteolytically decomposed and rapidly eliminated; thus, a steady and high GnRH analogue level, required for development of a direct or indirect antitumour effect, cannot be achieved. The retention of a high and steady level required for the inhibition of the tumour can be solved by a very frequent administration (daily several times) or by means of pharmaceutical compositions with prolonged effect. Up to the present, the microcapsule or microgranular form of GnRH analogues has been used for tumour inhibition in the clinical practice (Buserelin retard(copyright) and Decapeptyl retard(copyright) agonistic compositions).
According to another method to achieve a prolonged effect the pharmacologically active molecule is chemically coupled to molecules (e.g. polymers) slowly eliminating from the body. Conjugates thus obtained slowly diffuse in biological systems and alter the distribution in the body and absorption properties of the active agent. Thus, targeted transport of the pharmacologically active molecule and the reduction of undesired side effects can also be attained.
Preferred carriers are the water-soluble native and synthetic polymers, first of all homo- or copolymers, respectively, of carboxylic acids as synthetic polymers. Styrene/maleic anhydride copolymer- has a molecular weight of about 1600 to 2000 D, consisting of 7 to 8 styrene/maleic anhydride units wherein maleic anhydride had partially been hydrolyzed or esterified, respectively, was successfully used for improvement of the pharmacological properties of neo carcinostatin. In this way a more favourable distribution in the body together with a reduction of toxicity of the active agent could be achieved [H. Maeda et al.: J. Med. Chem. 28, 455-461 (1985)].
Polyanionic macromolecules with higher molecular weight may also possess themselves biological activity (e.g. antitumour, immunoadjuvant, interferon-inducing effects). However, their molecular weight and the distribution of the molecular weight proved to be crucial, i.e. their toxicity was increased by the increase in the molecular weight [L. Gros: Encyclopedia of Polymer Science and Technology, Vol. 2, pp. 243-267; as well as G. Butler: J. Macromol. Sci. Chem. A13, 351-368 (1979); and R. Ottenbrite: ibid. A22, 819-832 (1985)].
Thus, such polymers can be considered as carrier molecules that can be prepared with a good reproducibility, small polydispersity, which are not toxic and are eliminated at an optimum rate from the body. It is also important that the pharmacologically active molecules be capable of being coupled with a good reproducibility to these polymers to obtain a product with an appropriate solubility in water and the desired pharmacological activity.
Up to the present, peptide hormones and within these GnRH or GnRH analogues have not been coupled to known polycarboxylic acids as carriers. Copolymers prepared from vinylpyrrolidone and maleic anhydride are described in the Hungarian patent specification No. 194,286 (Hungarian patent publication No. 34519). These copolymers were prepared for cosmetical use; they are very useful skin-cosmetics and can be employed both in hydrophilic as well as lipophilic type emulsions. Depending on the amount of the base added, the pH value of their aqueous solutions can be varied within wide limits, they possess a high buffer capacity and can be prepared with a narrow range of polydispersity and with a good reproducibility. The vinylpyrrolidone and maleic anhydride units are incorporated to the molecule in a 1:1 molar ratio, with an alternating sequence.
Earlier, the toxicity, elimination and body distribution of the N-vinyl-pyrrolidone/maleic acid copolymer (NVP-MA) had been investigated in detail and the following statements were made [M. Azori et al: Macromol. Chem. 187, 297-302 (1986)]. On the intraperitoneal or intravenous, respectively, administration of the polymer (with an average molecular weight of 20,000) in the form of its sodium salt of pH 7.2, no death occurred up to 900 mg/kg of body weight in the first case (i.p.) or up to 200 mg/kg of body weight dose, respectively, in the second case (i.v.). This indicates that NVP-MA is less toxic than many other polyanions having a similar structure. [The intravenous (i.v.) LD50 value of a divinyl ether/maleic acid copolymer with similar molecular weight is 74 mg, that of polymaleic acid is 110 mg and that of furan/maleic acid copolymer amounts to 130 mg.] Body distribution examinations carried out with a 14C-labelled polymer (having an average molecular weight of 8000) indicated that the polymer cannot enter the brain and spinal cord. The polymer is eliminated mainly in the urine: 84% of the radioactivity introduced were eliminated during 24 hours whereas after 56 hours altogether 95% of the radioactivity introduced could be detected in the urine and feces. NVP-MA and its known derivatives until now have not been used in the therapy but on the basis of their favourable properties it seems possible to introduce them into the body without toxic effects.
The prior art is still deficient in the lack of effective means of inhibiting the wide variety of neoplastic conditions. The present invention fulfills this longstanding need and desire in the art.
The invention relates to novel, pharmacologically active compounds of general formula (I), their salts and complexes as well as to a process for preparing same. In the general formula (I)
Y(Wu,Vz,Xr,Ak)xe2x80x83xe2x80x83(I)
Y means the molecular moiety of general formula (Ia), 
wherein n is an integer from 10 to 400, preferably 20 to 200; one of R1 and R2 stands for hydrogen whereas the other one means a group of formula (B), 
R3 means a polymeriation-initiating group, preferably (CH3)2CCN group;
W means a hydroxyl group, optionally as a salt formed with an alkali metal ion, preferably sodium ion;
V represents a C1-8, preferably C4-6, alkylamino group bonded through its amino group; or a valence bond;
X means an amino acid group or an oligopeptide group of at most six members wherein the amino acid or oligopeptide group is coupled through its N-terminal to the Y group and is optionally bearing a hydroxyl group or a valence bond on its C-terminal, wherein the amino acids are Gly, Ala, Leu, Ile, Val, Phe, Tyr, Ahx, Pro, Arg or His;
A is present and represents a pharmacologically active polypeptide hormone group containing an amino group and directly coupled therethrough to the Y group when r is 0; or coupled to the C-terminal of the X group, respectively,
when r is larger than 0;
r is an integer from 0 to 0.2 n;
k is an integer being at most equal to r;
z is an integer from 0 to (nxe2x88x92r); and
u is an integer from n to 2nxe2x88x92rxe2x88x92z, as well as the salts and complexes of these compounds.
The invention furthermore relates to the novel intermediates of general formula (Ic),
Y[Wu, Vxe2x80x2z, (XOQ)r]xe2x80x83xe2x80x83(Ic)
wherein
Y means the molecular moiety of general formula (Ia), wherein n is an integer from 10 to 400, preferably 20 to 200; one of R1 and R2 stands for hydrogen atom whereas the other one means a group of formula (B); 
R3 means a polymerization-initiating group, preferably (CH3)2CCN group;
W means a hydroxyl group, optionally as a salt formed with an alkali metal ion, preferably sodium ion;
Vxe2x80x2 stands for a C1-8, preferably C4-6, alkylamino group bonded through its amino group;
X represents an amino acid group or an oligopeptide group of at most six members coupled through its N-terminal to the Y group;
OQ means an activated ester group on C-terminal of the X group, preferably
ONp, OPcp, OPfp or ONSu group;
r is an integer from 0 to 0.2 n;
z is an integer from 0 to (nxe2x88x92r); and
u is an integer from n to (2nxe2x88x92rxe2x88x92z), as well as the salts of these compounds.
Furthermore, the invention relates to tumour-inhibiting and immunostimulatory pharmaceutical compositions comprising as active ingredient a compound of general formula (I). The bioconjugates of general formula (I) possess a selective tumour-inhibiting effect; a part of the compounds of general formula (I) inhibit the growth of both steroid-dependent and steroid-independent tumours, particularly mammary carcinomas. The compounds of general formula (I) show the effect of the pharmacologically active moiety in an increased and prolonged degree.
The invention relates to novel peptides possessing antitumour effect as well as their salts and esters. In addition to antiestrogens, gonadotropin-releasing hormone (GnRH) analogues play an important role in the treatment of hormone-dependent malignant tumours. Within the malignant neoplasms, the scope of their use extends to the cancers of prostate, breast (mammary), endometrium and other hormone-dependent tumours. The present invention prepares analogues of human GnRH (hGnRH) and Lamprey GnRH-III showing antitumour effect in human tumour cell cultures. This aim was solved by the preparation of peptides of general formula (IV) as well as their pharmaceutically acceptable salts and esters.
The invention is based on the recognition that these compounds exert a direct antitumour action against human tumour cells. Unexpectedly, compounds containing only natural L-amino acids also show a direct antitumour effect. Namely, antitumour GnRH analogues known at present contain at least one of non-natural D-amino acids in the case of agonists and usually several non-natural D-amino acids in the case of antagonists.
Furthermore, it has been recognized that for more amino acid groups of antitumour GnRH analogues Lys groups may be substituted without any decrease in the favourable antitumour effect of the molecule. This is advantageous since, through the e-amino group of Lys, the peptide can be connected to suitably selected larger molecules containing an acylating group. In this case, the macromolecules may be carrier molecules of the peptide and can thereby promote the maintenance of a steady, high level of the GnRH analogue in the body.
The invention relates to the peptides of general formula (IV),
X-R1-R2-R3-R4-R5-R6-R7-R8-Pro-R10-Yxe2x80x83xe2x80x83(IV)
wherein
X means hydrogen, acetyl group or propionyl group when R1 is different from pGlu; or an intramolecular acid amide bond when R1 stands for pGlu;
R1 stands for pGlu, Glu, D-Trp, D-Cpa, D-Nal or D-Phe;
R2 means His, D-Phe or D-Cpa;
R3 represents D-Cpa, D-Pal or L- or D-Trp optionally protected on the indolyl moiety;
R4 stands for Ser; or Lys optionally protected on the xcex5-amino group;
R5 means Tyr; or Lys optionally protected on the xcex5-amino group; or His;
R6 stands for Asp, Glu, D-Lys and optionally xcex5-amino methylated derivatives thereof; as well as D-Trp, D-Phe, D-Leu, D-Ala, D-Cpa or D-Arg;
R7 represents Phe, Leu or N-Me-Leu; or L-Trp optionally protected on the indolyl moiety;
R8 means Lys optionally protected on the xcex5-amino group; Arg, Gln; or R6 and R8 together can form an intramolecular ring through the xcex5-amino group of Lys when R6 is Asp and R8 means Lys;
R10 stands for Gly, D-Ala or a valence bond; and
Y represents OH or NH2 group when R10 means Gly or D-Ala; or an ethylamide group when R10 means a valence bond, as well as the pharmaceutically acceptable salts and/or esters of these compounds. The invention furthermore relates to pharmaceutical compositions containing the peptide of general formula (IV) and/or pharmaceutically acceptable salts and/or esters thereof.
The abbreviations used in the description agree with the nomenclature accepted in the peptide chemistry and published in Edsall et al., eds., J. Biol. Chem. 241, 527-533 (1966); Tabor et al., eds., J. Biol. Chem. 247, 977-983 (1972); furthermore, D-Nal stands for xcex2-(2-naphthyl)-D-alanine, D-Cpa means p-chlorophenyl-D-alanine and D-Pal stands for xcex2-(3-pyridyl)-D-alanine. When not noted otherwise, all amino acids named in the description are in L-configuration.
In the peptides according to the invention the preferred protective group of the indolyl moiety of Trp is For; the preferred protective group of the C-amino group of Lys is Fmoc. The pharmaceutically acceptable salts of the peptides of general formula (IV) are acid-addition salts formed with pharmaceutically acceptable organic or inorganic acids, e.g. acetates or hydrochlorides.
The compounds of general formula (IV) can be prepared in liquid phase by using methods known in the peptide chemistry (by condensations carried out in the defined sequence of suitably protected amino acids or fragments prepared therefrom) or in a particularly preferable wayxe2x80x94by using the solid-phase peptide synthesis. A peptide obtained in the form of its salt can be convened to an other salt in a known manner. If desired, the ester groups of ester compounds obtained may be cleaved.
The peptide of general formula (IV) may be administered mainly in the form of injectable solutions, infusions or intranasal compositions. Being decomposed in the digestive system, they cannot be administered orally in themselves but may be administered in any other route. The injections may be given in intramuscular, intravenous or subcutaneous route.
The active agents of general formula (IV) can be formulated to pharmaceutical compositions by using known methods of the pharmaceutical techniques. The active agent can be transformed also to compositions with prolonged action (e.g. in the form of microcapsules or microgranules) in the usual way. In addition to the active agent, auxiliaries commonly used in the pharmaceutical industry such as a liquid vehicle useful for injection purposes (isotonic saline or phosphate buffer solution) may be used. If necessary, the compositions may contain stabilizers (e.g. ascorbic acid), too.
The preparation of peptides according to the invention is illustrated by the following Examples. The chemical purity and identification of both intermediary and final products were controlled by using thin-layer chromatography (TLC); those of the final products were examined by means of HPLC too. The thin-layer chromatography values were determined on Kieselgel sheets (DC Alufolien, Merck) by using the following solvent mixtures:
1. Ethyl acetate/pyridine/water/acetic acid 15:20:6:11
2. Ethyl acetate/pyridine/water/acetic acid 30:20:6:11
3. Ethyl acetate/pyridine/water/acetic acid 60:20:6:11
4. Ethyl acetate/pyridine/water/acetic acid 120:20:6:11
5. Ethyl acetate/pyridine/water/acetic acid 240:20:6:11
6. n-Butanol/acetic acid/water 4:1:1
7. n-Butanol/acetic acid/water 4:1:2
The side chains of protected amino acids are protected by a benzyl group in the case of Tyr and Ser; by a (benzyloxy)carbonyl (Z) group or a 9-fluorenyl(methoxycarbonyl) (Fmoc) group for preparing an intermediary peptide analogue in the case of Lys; by a tosyl (Tos) group in the case of Arg and His; and by a cyclohexyl (Chx) group in the case of carboxy groups of Asp and Glu.
The invention is illustrated in more detail by description of the preparation process of the preferred analogues listed hereinbelow:
1. [Lys(xcex5-Fmoc)]5-GnRH-III,
2. Lys5-GnRH-III,
3. Lys5,cyclo[Asp6-Lys8]-GnRH-III,
4. Lys5,[Lys(xcex5-Fmoc)]8-GnRH-III,
5. Lys4,[Lys(xcex5-Fmoc)]8-GnRH-III,
6. Lys4-GnRH-III,
7. [Lys(e-Ac)]4-GnRH-III,
8. Glu6-GnRH-III,
9. cyclo[Asp6-Lys8]-GnRH-III,
10. D-Ala10-GnRH-III,
11. H-D-Trp1, [Lys(xcex5-Fmoc)]8, D-Ala10-GnRH-III,
12. Ac-D-Trp1, D-Ala10-GnRH-III,
13. H-D-Trp1, D-Ala10-GnRH-III,
14. [Trp(For-Ind)]3,7-GnRH-III,
15. Phe7-GnRH-III,
16. GnRH-III(1-9)-ethylamide,
17. Lys5, D-Trp6-hGnRH,
18. Lys4, D-Trp6-hGnRH,
19. H-Glu1, D-Trp-hGnRH,
20. Lys5, D-Phe6-hGnRH(1-9)-ethylamide,
21. Lys4, D-Phe6-hGnRH(1-9)-ethylamide,
22. Lys5, D-Cpa6-hGnRH(1-9)-ethylamide.
On investigating the capacity factor of some analogues of high importance by using HPLC the following results were obtained:
ISCO model 2350 pump 1 ml/min, ISCO V4 detector (215 nm). Column: BST, ODS Hypersil 5 xcexcm, 270xc3x974 mm. Eluent: MeOH/0.1 M NaH2PO4 (pH=2.22).       k    xe2x80x2    =                    T        R            -              t        0                    t      0      
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
The abbreviations used in the description agree with the nomenclature accepted in the peptide chemistry and published in Edsall et al., eds., J. Biol. Chem. 241, 527-533 (1966); and Tabor et al., eds., J. Biol. Chem. 247, 977-983 (1972); furthermore, D-Nal stands for a-(2-naphthyl)-D-5-alanine, D-Cpa means p-chlorophenyl-D-alanine and D-Pal stands for a-(3-pyridyl)-D-alanine. When not noted otherwise, each of the amino acids mentioned in the description is in L-configuration.
The new compounds of general formula (I) can be prepared by coupling pharmacologically active polypeptides, preferably GnRH analogues, to molecules of general formula (Ia) in the following way: a) pharmacologically active compounds containing a free amino group are coupled to N-vinylpyrrolidone/maleic anhydride copolymer of general formula (Ib) 
known per se but not used in the therapy up to the present, wherein R1 R2 and R3 are as defined for the general formula (Ia); b) a compound of general formula (Ib) is reacted with an activated ester of general formula (IR)
H-X-OQxe2x80x83xe2x80x83(III)
of an amino acid or an oligopeptide, then the pharmacologically active polypeptides are coupled to the carrier compound of general formula (Ic) obtained by means of the activated ester groups. The compounds obtained can be transformed to salts of a pH value of 7.2, preferably to sodium salts.
The pharmacologically active molecule (A) appearing in the general formula (I) is preferably an agonistic or antagonistic GnRH analogue. The X group of compounds of general formula (I) means an amino acid group or an oligopeptide group of at most six members built up from native or non-natural amino acids. These can be prepared by using processes known in the peptide chemistry.
The invention is based on the following recognitions:
1. In comparison to the corresponding pharmacologically active peptide hormones, chiefly GnRH analogues, in relation to the original effect of the peptide hormone molecules, the compounds of general formula (I) show an increased action prolonged in time.
2. Compounds of general formula (I) containing a spacer group as X, i.e. wherein r is different from 0, show an even more favourable effect than those compounds containing no X group. Simultaneously, no toxic side effects are shown by the compounds containing X group or those without X group; and these compounds retain all effects of the pharmacologically active polypeptide molecule A.
3. Compounds of general formula (I) and their salts, wherein A represents a GnRH analogue of formula (IIa) coupled through the amino group of the side chain of the Lys group of the peptide hormone, Y, W, V, X, r, k, z and u are as defined for the general formula (I), are tumour-inhibiting compounds retaining and even exceeding the known pharmacological effects of the analogue (Ha), i.e. pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2 (SEQ ID NO:3); MI-1544: Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10; thus, they inhibit the growth of both steroid-dependent and steroid-independent tumours. In addition, they induce a reversible chemical castration enhancing the antitumour action in the case of steroid-dependent tumours.
4. Compounds of general formula (I), wherein A stands for a GnRH antagonist analogue of formula (IIb), i.e., MI-1892: Ac-D-Trp1,3,D-Cpa2,Lys5, [Asp(a-DEA)]6,D-Ala10-Gln8-GnRH coupled through the n-amino group of lysine in position 5, and Y, W, V, X, r, k, z as well as u are as defined for the general formula (I), are selective tumour-inhibiting agents according to their antagonistic character; thus, they inhibit the growth of both steroid-dependent and steroid-independent tumours, actually to a degree surpassing the effect of the compound of formula (IIb). Both the antagonist of formula (IIb) and its derivative of general formula (I) possess an irreversible chemical castration effect which, together with the selective direct tumour-inhibiting effect, is preferred from the view-point of effectivity of tumour inhibition. It has unexpectedly been observed that the antagonist of formula (IIb) possesses also an effect stimulating the immune system, and this effect is retained by the derivative of general formula (I), too. This recognition is surprising, since the therapeutic use of tumour-inhibiting GnRH antagonists known up to now has been impeded just by their immunosuppressive action.
5. Compounds of general formula (I), wherein A stands for the lamprey GnRH-III coupled through the n-amino group of the lysine moiety in position 8, whereas Y, W, V, X, r, k, z and u are as defined for the general formula (I), are selective tumour-inhibiting agents under in vivo conditions and therefore, they inhibit the growth of both steroid-dependent and steroid-independent tumours in spite of the fact that the GnRH-III alone shows only a negligible in vivo effect. Conjugates of general formula (I) of GnRH-III, exceeding the effects of sustained-release hGnRH compositions, resulted in a tumour-free state of the experimental animals in a treatment period, where only a decrease in the tumour growth was observed by using known compositions. The most preferable compounds of general formula (I) according to the invention were prepared by conjugating the following GnRH analogues of formulae (IIa) to (IIi): human GnRH (hereinafter GnRH):
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO:4); chicken GnRH (hereinafter Gln8-GnRH):
pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2 (SEQ ID NO:2); lamprey GnRH-III (hereinafter GnRH-III):
pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2 (SEQ ID NO:3); MI-1544: Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10-GnRH antagonist analogue of formula (IIa) of human GnRH;
MI-1892: Ac-D-Trp1,3,D-Cpa2,Lys5,[Asp(a-DEA)]6,D-Ala10-Gln8-GnRH; antagonist analogue of formula (IIb) of chicken GnRH;
SJ-1004: D-Phe2,D-Trp3,D-Lys6-GnRH; antagonist analogue of formula (IId) of human GnRH;
TH-614: Lys5,cyclo(Asp6-Lys8)-GnRH-III; analogue of formula (IIe) of lamprey GnRH-III;
HB-694: Lys4,[Lys(n-Fmoc)]8-GnRH-III; analogue of formula (IIf) of lamprey GnRH-III;
TH-602: Lamprey GnRH-III; see above the formula; GnRH of formula (IIc);
HB-685: Lys4-GnRH-III; analogue of formula (IIg) of lamprey GnRH-III;
TH-609: D-Lys6-GnRH; analogue of formula (IIh) of human GnRH;
TH-615: Lys5,D-Trp6-GnRH; analogue of formula (IIi) of human GnRH.
The GnRH analogues were coupled either directly or through an X group, i.e. spacer moiety, to the polymer designated as P. For naming the spacers, the one-letter marking of the amino acids are used, e.g. H-GFLG-OH: H-Gly-Phe-Leu-Gly-OH (SEQ ID NO:5) (peptide in the form of its sodium salt); P: polyvinylpyrrolidone/maleic acid copolymer sodium salt, i.e. a salt of formula (Id), wherein one of R1 and R2 means hydrogen whereas the other one is a group (B), n is 66, transformed to the sodium salt by n equivalents of NaHCO3.
The biological study of the following substances was carried out: P-GLG-1892: compound of general formula (I), wherein A=(IIb) group coupled to the Y=(Ia) group through X=xe2x88x92GLG-moiety; n=66, r=0.1n, k=0.3r, z=0, u=1.9n and W=OH or ONa, respectively, pH=7.2;
P-GFLG-1544: compound of general formula (1), wherein A=(IIa) group coupled to the Y=(Ia) group through the X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r, z=0, u=1.9n and W=OH or ONa, respectively, pH 7.2;
P-1892: compound of general formula (I), wherein A=(IIb) group, n=66, r=0, k=0.024n, z=0, u=1.9n, W=OH or ONa, respectively, pH 7.2;
P-GFLG-1892: compound of general formula (I), wherein A=(IIb) group coupled to the Y=(Ia) group through an X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r, z=0, u=1.9n and W=OH or ONa, respectively, pH 7.2;
P-GFLG-609: compound of general formula (I), wherein A=(IIb) group, coupled to the Y=(Ia) group through an 20 X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH=7.2;
P-GFLG-1004: compound of general formula (I), wherein A=(IId) group, coupled to the Y=(Ia) group through an 25 X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH 7.2;
P-GFLG-614: compound of general formula (I), wherein A=(IIe) group, coupled to the Y=(Ia) group through an X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH=7.2;
P-GFLG-685: compound of general formula (I), wherein A=(IIg) group, coupled to the Y=(Ia) group through an X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH=7.2;
P-GFLG-602: compound of general formula (I), wherein A=(GnRH-III) group, coupled to the Y=(Ia) group through an X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH 7.2;
P-GFLG-615: compound of general formula (I), wherein A=(IIi) group, coupled to the Y=(Ia) group through an X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH=7.2;
P-FLG-892: compound of general formula (I), wherein A=(IIb) group, coupled to the Y=(Ia) group through an X=xe2x88x92GFLG-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH=7.2;
P-Ahx-1892: compound of general formula (I), wherein A=(IIb) group, coupled to the Y=(Ia) group through an X=xe2x88x92Ahx-moiety; n=66, r=0.1n, k=0.3r; z=0; u=1.9n and W=OH or ONa, respectively, pH 7.2.
The biological tests were carried out by using the materials and methods following hereinafter. Certain human mammary carcinoma cell lines or xenografts prepared therefrom are useful for the biological testing of the conjugates.
Human cell lines used in in vitro experiments. MCF-7 human mammary tumour cell line was stabilized in 1973 by Soule et al. [J. National Cancer Inst., 51, 1409-1416] from the pleural fluid of mammary carcinoma patients. The cells grow in monolayer and are epithelial in their character. MDA-MB-231 human mammary carcinoma cell line was isolated and stabilized in 1974 by Cailleau et al. [J. National Cancer Inst., 53, 661-674] similarly from pleural fluid. These cells also grow in monolayer. PC3 human prostate carcinoma cell line was stabilized 20 in 1979 in cell culture by Kaighn et al. [Investigative Urology, 17, 16-23]; the cells are of simple epithelium type and form compact colonies in clonogenic assays. Ishikawa cell line originated from adenocarcinoma of human endometrium [Nishida et al.: Obstet. Gynecol. Jpn, 37 1103-1111 (1985)], has epithelial character and contains steroid as well as GnRH receptors. Human tumour cell lines are maintained in plastic flasks (Greiner) in Dulbecco-modified Eagle-MEM (DMEM GIBCO) nutritive medium. The medium used herein contained 10% of fetal calf serum (FCS).
The MCF-7 cell line is estradiol-receptor (ER) positive and GnRH receptor-positive. Therefore, it is suitable to study the receptor-mediated direct effect of GnRH; being an in vivo model system, it is useful for investigating both the direct and indirect effects of GnRH. Being an ER-negative and GnRH receptor-positive in vitro and in vivo model system, MDA-MB-231 cell line is useful to study the direct effect of GnRH. Based on literature data, PC3 human prostate carcinoma cell line contains receptors specifically binding GnRH, whereby the essential condition for the direct effect is fulfilled. Ishikawa cell line is originated from human endometrium adenocarcinoma, contains ER and PgR proteins and is GnRH receptor-positive. Thus, it is useful for investigating the direct effect of GnRH. Buserelin, chemically [D-Ser(tBu)]6,desGly10-hGnRH(1-9)EA, is a tumour-inhibiting agonist being used in a retard form in clinical practice; conclusively, the substance itself but not its retard form was employed in the in vitro experiments.
The preparation of the known polymeric carrier and the investigation of compounds of general formula (I) as well as of the spacers of general formula X were carried out as follows.
NVP-MA samples used herein were prepared according to the literature [Reddy et al.: Polymer 25, 115-120 (1984)]. Activated esters of oligopeptides employed as spacers were preferably nitrophenyl esters obtained by using classic methods of peptide chemistry. The coupling of peptides to polymers can be accomplished in solvents, such as dimethylsulfoxide (DMSO), dimethylformamide (DMF) or dimethylacetamide. DMF was used as solvent.
Nitrophenyl esters of peptides were characterized by amino acid analysis. The retention factors referred to were determined on Kieselgel 60 F254 thin layer by using the following developing systems:
1. pyridine:acetic acid:water:ethyl acetate 20:6:12:62;
2. chloroform:methanol:water 10:5:1.
High pressure liquid chromatography (HPLC) examinations were carried out on a C-18 reverse-phase column at a flow rate of 0.5 ml/min by using the following gradients:
Eluent A: 5% of acetonitrile plus 95% of triethylamine-phosphate buffer of pH 2.25;
Eluent B: 80% of acetonitrile plus 20% of triethylamine-phosphate buffer of pH 2.25; 100% of eluent A up to the 5th minute; to 60% of eluent A up to the 10th minute; to 35% of eluent A up to the 30th 25 minute; to 0% of eluent A up to the 35th minute; 0% of eluent A up to the 40th minute; and to 100% of eluent A up to the 45th minute. The detection was performed at 280 nm with an UV detector. The conjugates were purified by ultrafiltration on AMICON PM10 membrane.
The results advantages of the new compounds can be summarized as follows. Based on the in vitro examinations it can be stated that the proliferation- and colony formation-inhibiting effects of the conjugates P-GFLG-1544 and P-GFLG-1892xe2x80x94containing the GFLG tetrapeptide spacer groupxe2x80x94highly exceed the antitumour effect of both the GnRH analogue substances (i.e. MI-1544 or MI-1892) as well as that of the spacer-containing carrier (P-GFLG-OH) and even that of the compound P-1892. The conjugates (i.e. compounds built up from the carrier and GnRH analogue) resulted in colony formation-inhibition of 100% above a concentration of 20 xcexcM. Such a high grade of inhibition could until now be achieved only by the use of cytostatics. Based on the results both the polymeric starting substance and the spacer-containing polymer as a polyanion in themselves also exert a direct antitumour effect, and, when coupled covalently to GnRH antagonists, they enhance the direct antitumour action of the antagonists.
According to the in vivo studies the conjugates P-GFLG-1544 and P-GFLG-1892 as well as the carrier P-GFLG-OH proved to be non-toxic. The conjugates P-GFLG-1544 and P-GFLG-1892 resulted in a tumour volume diminution of 30 to 35% in the second week and 37 to 49% in the fourth week of treatment on the ER-positive, GnRH receptor-positive MCF-7 human mammary carcinoma xenograft; whereas a tumour volume diminution of 37 to 42% could be observed on the ER-negative, GnRH receptor-positive MDA-MB-231 human mammary carcinoma xenograft. This result is considered to be significant since the degree of tumour inhibition observed by us on human carcinoma xenograft is nearly identical with that of the sustained-release composition of SB-75, a GnRH antagonist [Szepeshazi et al.: Breast Cancer Res. Treat. 21, 181-192 (1992)], which had been achieved on the MXT mouse mammary carcinoma which is more sensitive.
The substance MI-1892 and its conjugate P-GFLG-1892 possess a direct, selective tumour-inhibiting effect. Namely, in contrast to substance MI-1544 and its conjugate P-GFLG-1544, they exert a minimum chemical castration effect. Based on this fact it can be supposed that they act on a broader spectrum of mammary carcinomas and therefore, on hormone-(estrogen-)-independent tumours, too.
When investigating the effect of the compounds on the cellular and humoral immune system it was stated that MI-1892 as selective chicken GnRH antagonist as well as the conjugate P-GFLG-1892 containing the antagonist and the spacer-containing P-GFLG, a novel polyanionic macromolecule, increased the activity of T-lymphocytes against bovine red blood cell (BRBC) antigen (by using the method of rosette formation). On the basis of examinations of the antibody production of B-lymphocytes, MI-1892 and P-GFLG-OH as well as the conjugate P-GFLG-1892 containing MI-1892 moiety strengthened the humoral immune response, too.
The immunosuppressive side effect of cytostatics used in tumour therapy is commonly known. This effect can be compensated to a certain grade by various immunostimulants (endotoxin, levamisole). Polypeptides with much lower molecular weights (about 40,000 to 80,000) are also capable of protecting against immunosuppressive effect [Gaxc3xa1l et al.: J. Biol. Resp. Modif. 5, 148-159 (1986)]. The molecular weight of the novel polyanionic P-GFLG-OH macromolecule tested by us is about 10,000, that of MI-1892 is about 2000, i.e. relatively low; therefore, their immunostimulatory activity was unexpected. The importance of these results is reflected by the fact that MI-1892 administered to mice in a dose of 50 xcexcg/mouse or 100 xcexcg/mouse, respectively, increased the humoral immune response to more than twofold or threefold, respectively; the increase was twofold in the case of P-GFLG-OH. The conjugate P-GFLG-1892 also possesses an immunostimulatory effect. The investigation of the cellular immune response showed that MI-1892 given in a dose of 100 xcexcg/mouse increased the response to nearly twofold; this increase was twofold in the case of P-GFLG-OH and P-GFLG-1892.
The novel polycarboxylic acid derivatives according to the invention are very preferable carriers for peptide hormones since they can be prepared with a good reproducibility and with a low polydispersity in the aimed range of molecular weights and they are water-soluble.
Known polycarboxylic acid derivatives also possess a weak tumour-inhibiting action and can similarly favourably be used as carrier compounds. By coupling pharmacologically active compounds such as GnRH analogues to the polymers of the invention, novel compounds are obtained which exert an increased therapeutic efficiency in comparison to that of their structural moieties.
The new conjugate P-GFLG-1544 is a compound with favourable therapeutic effect for the following reasons: It has a selective tumour-inhibiting effect proved by the direct inhibition of cell proliferation. Thus, it inhibits not only the growth of steroid-dependent but also that of steroid-independent tumours as demonstrated by investigations carried out on MDA-MB-231 mammary tumour cells. Due to the antagonistic effect, the coupled MI-1544 possesses a reversible castration effect. This effect is proved by the change of uterus weight measured during in vivo treatments as well as the decrease in the cytosolic progesterone receptor (cPgR) level of uterus. A very important advantage of this effect is that the hormone status is re-established by suspending the treatment. This is opposed to the chirurgical intervention and irradiation, which are irreversible and refused by many patients. The advantage of castration effect is that in the case of steroid-dependent tumours the selective direct and castration effects are summarized to result in a stronger inhibition of the tumours.
The castration effect is illustrated by the following data. After a treatment for four weeks the uterus weights of animals bearing MCF-7 xenografts were decreased by 42% (control=0.1980 q 0.0120 g; P-GFLG-1544=0.1149+0.0110 g); after a treatment for twelve weeks the uterus weights of animals bearing MDA-MB-231 xenografts were decreased by 50% (control=0.0263+0.0028 g, P-GFLG-1544=0.0133 q 0.0018 g). The weight of uterus was not decreased but slightly increased by P-GFLG-OHalone (0.0337 q 0.0034 g, 128%). The change of progesterone level was proved by the significant decrease (of 54%) of the uterus cPgR level of animals bearing MCF-7 xenografts (control=517 q 45 femtomol/mg of protein, P-GFLG-1544=241 q 28 femtomol/mg of protein).
In opposition to several known GnRH antagonists, P-GFLG-1544 does not possess immunosuppressive effect. Summing up: the conjugate P-GFLG-1544 not only retained but also substantially exceeded both the selective tumour-inhibiting as well as the castration effects of the MI-1544, an antagonist analogue of GnRH, mainly in respect of tumour inhibition. No adverse side effects have been observed during the investigations on the conjugate. Due to its prolonged effect this novel antagonist compound can be utilized for in vivo tumour inhibition. The new conjugate P-GFLG-1892 is a favourable compound with therapeutic effect for the following reasons: It has a selective tumour-inhibiting effect. Therefore, it inhibits the growth of both steroid-dependent and steroid-independent tumours, which was proved by investigations on MDA-MB-231 cells.
The GnRH antagonist MI-1892 and the conjugate containing the antagonist possess a weaker castration effect in comparison to MI-1544 and its conjugate. The castration effect of the conjugate was demonstrated by the change in uterus weight measured during in vivo treatments as well as by the decrease in the cytosolic progesterone receptor (cPgR) level of uterus. After a treatment for four weeks the uterus weight of animals bearing xenograft was decreased by 39% (control 0.1980 q 0.0120 g, P-GFLG-1892=0.1215 q 0.0130 g). After a treatment for twelve weeks the uterus weight of animals bearing MDA-MB-231 was decreased by 20% (control=0.0263 q 0.0028 g, P-GFLG-1892=0.0215 q 0.0017 g). The weight of uterus was not decreased but slightly increased by P-GFLG-OH alone (0.0337 q 0.0034 g, 128%). The change in the progesterone level was proved by the 23% decrease in the uterus cPgR level of animals bearing MCF-7 xenograft (control=517 q 45 femtomol/mg of protein, P-GFLG-1892=376 q 33 femtomol/mg of protein).
In opposition to several known GnRH antagonists, it has no immuno-suppressive effect. It is a new recognition that this novel GnRH antagonist analogue and the conjugate containing this analogue possess immunostimulatory effect as proved by the humoral and cellular immune response investigations.
On comparison to healthy individuals, the functioning of the protective mechanism (immunostatus) of patients suffering from tumour is more unfavourable. Since the protective (immune) mechanism is enhanced by the compound, its tumour-inhibiting action can more strongly become valid. The novel conjugate P-GFLG-GnRH-III is a compound possessing favourable therapeutic action for the following reasons: It has a selective tumour-inhibiting effect.Therefore, it inhibits the growth of both the steroid-dependent and steroid-independent tumours which was proved by investigations on MDA-MB-231 cells. It has no castration effect since the cycle of female rats is not influenced even by a high dose of the coupled GnRH-III during three cycles observed. This effect may particularly be preferred on young patients suffering from mammary tumour, where a castration would cause psychic disturbances. In a way different from other conjugates, P-GFLG-GnRH-III gradually induces an inhibition of continually increasing grade during the long-lasting treatment for 7 weeks; and at the end of treatment, tumour-free animals are observed whereas in the case of other conjugates the degree of inhibition increases only up to the fifth week of treatment, then a stagnation in the grade of inhibition can be observed.
The pharmaceutical compositions containing the compound of general formula (I) according to the invention may be prepared by transforming the compound of general formula (I) or a pharmaceutically acceptable salt or complex thereof to a composition with carriers and/or additives commonly used in the pharmaceutical industry by using known operations of the pharmaceutical techniques.
The pharmaceutical composition for therapeutic use may contain any filling material and carrier used in the therapy (e.g. calcium carbonate, talc); solvent (such as water, an aqueous solution containing ethanol and/or polyalcohol, e.g. polyethylene glycol and/or glycerol and the like); salts (e.g. sodium chloride for adjusting the physiological osmotic pressure; or e.g. chlorides of iron, cobalt, zinc or copper and the like for supplementing trace elements); solubilizing additives, e.g. complex-forming agents (cyclodextrins, crown ethers, native proteins, saponins and the like); compounds diminishing the relative permittivity of the solvent such as ethanol, polyols (polyethylene glycol or glycerol); tablet-disintegrating agents; complex-forming agents commonly used in sustained-release compositions (e.g. water-insoluble cyclodextrin derivatives, native and artificial polymers, crown ethers and the like); pH-adjusting compounds such as mineral and organic buffers; taste-improving agents (beet-sugar, fructose and dextrose, saccharins, inverted sugar and the like); antioxidants (e.g. vitamin C); as well as other active ingredients promoting the effectuation of the action of active agents of general formula (I). The pharmaceutical compositions may be oral such as tablets, pills, drages, hard or soft capsules, microcapsules, solutions, emulsions or suspensions; or parenteral, e.g. injectable solutions, slow and rapid infusions; as well as pharmaceutical compositions useful for rectal administration such as suppositories; furthermore creams or jellies. There also exists the possibility of incorporating to liposomes the pharmaceutical compositions developed for the above uses. The bioconjugates of general formula (I) can be utilized also in aerosol compositions targeted at the absorption through the skin surface or the lungs, respectively. For the preparation of tablets, drages or hard gel capsules, e.g. calcium carbonate, talc, fats, waxes or polyalcohols, having an appropriate density, are useful carriers.
For the preparation of solutions and syrups, e.g. water, polyalcohols (e.g. polyethylene glycol or glycerol), beet-sugar or dextrose may be used as carriers. Parenteral compositions may contain water, alcohol, polyalcohols or vegetable oils as carriers. Carriers of suppositories may be e.g. oils, waxes, fats or polyalcohols having a suitable density. The bioconjugates of general formula (I) are useful for therapeutic utilization in combination with artificial and native active agents, too. The bioconjugates of general formula (I) are effective in a dose range of 0.01 to 100 xcexcg/kg in subcutaneous, intramuscular or intravenous injections. The dose to be used in the practice is dependent on the type of disease as well as on the state and age of the patient and it should be determined by the physician.